During fruit juice manufacture enzyme preparations are often used in the steps of extraction and liquefaction of fruit and fruit juice clarification (Voragen 1989). The commercial enzyme preparations contain a mixture of mainly pectinases (e.g. polygalacturonases, pectin esterases, pectin transeliminases) together with minor quantities of other hydrolytic enzymes such as arabinases, galactanases and xylanases. The substrates for the various pectinases are pectins, which are polygalacturonides of high molecular weight (20000-40000 D) consisting of .alpha.-1,4-glycosidic bound D-galacturonic acid polymers. Some of the uronic acid groups are esterified with methanol. The polygalacturonic backbone is interrupted by so-called hairy regions, consisting of a rhamnose-galacturonic acid backbone with arabinose-rich side chains (Voragen and Beldman 1990).
Pectins occur in nature as constituents of higher plant cell walls. They are found in the primary cell wall and middle lamella where they are embedded in cellulose fibrils (Mc Neil et al. 1984). The composition of pectin and the degree of methylation is variable among plant species and moreover dependent on the age and maturity of the fruit. Among the richest sources of pectins are lemon and orange rind, which can contain up to 30% of this polysaccharide.
Pectinases can degrade the carbohydrate polymer either by hydrolysis of the .alpha.-1,4-glycosidic bond (endo and exopolygalacturonases or by transelimination reaction (pectin lyases). Pectin esterases can demethylate highly esterified pectin into polygalacturonic acid. Pectin lyases are specific for highly esterified pectins, polygalacturonases hydrolyse low esterified pectins. Consequently highly esterified pectins can be degraded by pectin lyases or the combination of pectin esterases and polygalacturonases (Pilnik 1982).
In the various stages of fruit and vegetable processing pectinases play an important role. Originally pectinases were used for treatment of soft fruit to ensure high yields of juice and pigments upon pressing and to clarify raw press juices. Polygalacturonases are used as macerating enzymes for the production of pulpy nectars, loose cell suspensions that are the result of limited pectin breakdown particularly in the middle lamella. A combination of several pectinases together with cellulolytic enzymes is needed to almost completely liquefy fruit tissue, thereby facilitating extraction (Renard et al. 1989). The clarification of apple juices can for example be improved by the combined activity of pectin esterases and polygalacturonases or by pectin lyases for which the highly esterified apple pectin is an ideal substrate (Ishii and Yokotsuka 1973).
Most of the pectinases present in commercial preparations are of fungal origin. Aspergillus niger is the most important organism for the industrial production of pectin degrading enzymes. In A. niger the various pectinases are not expressed constitutively (Maldonado et al. 1989). Pectin or degradation products of the pectin molecule are needed as inducing substances. The fermentation conditions for pectinase production often result in a wide spectrum of pectinases. Moreover, A. niger produces many isoenzymes of the various pectinases. Recently patents have been published describing that genes encoding polygalacturonase (EPO 0421 919, EPO 0 388 593), pectin lyases (EPO 0 278 355, EPO 0 353 188) and pectin esterases (EPO 0 388 593) have been isolated and used for the construction of overproducing transformants. These transformants allow the production of specific enzymes, needed e.g. in maceration applications and in studies on the effect of the various pectinases in processes like liquefaction and clarification.
Schols et al. (1990b) have described the isolation and characterization of a cell-wall polysaccharide from apple juice obtained after the liquefaction process in which the juice was released from the apple pulp by the combined action of pectolytic and cellulolytic enzymes. These cell-wall polysaccharides resemble the hairy regions of apple pectin (a rhamnose-galacturonic acid backbone with arabinose rich side chains) and have been called Modified Hairy Regions (MHR). Hairy regions are known to be present not only in apples but also in carrots, grapes and strawberries and are probably a common part of pectin molecules. The modified hairy regions are resistant to breakdown by the enzymes present in most pure and technical pectinase and cellulase preparations. So only a commercial crude enzyme preparation obtained from Aspergillus aculeatus has been found to be able to depolymerize the rhamnogalacturon backbone of these fragments. This activity was made visible by measuring the shift in molecular weight distribution using High Performance Gel Permeation Chromatography (HPGPC). Schols et al. (1990a) purified the enzyme responsible for the degradation of the modified hairy regions prepared from apple juice and called the enzyme rhamnogalacturonase (RGase). The enzyme can split glycosidic linkages in the rhamnogalacturonan backbone of (apple) pectins producing, besides other not yet fully identified reaction products, a range of oligomers composed of galacturonic acid, rhamnose and galactose with rhamnose at the non reducing end, hence the name rhamnogalacturonase (RG-ase) for this novel enzyme. The oligomers present after incubation of MHR with rhamnogalacturonase were found to be a mixture of a tetramer (Rhamnose(2)-Galacturonic acid(2)) and a hexamer (Rhamnose(2)-Galacturonic acid(2)-Galactose(2) (Colquhoun 1990)). Schols et al (1990a) used various chromatographic steps and column materials to isolate and purify the enzyme with RG-ase activity. Rhamnogalacturonase was found to be inactive against MHR but was very active towards MHR-S and MHR-HCl. MHR-S is saponified MHR in which the methoxycarbonyl and acetyl groups have been removed. MHR-HCl is MHR from which the arabinan groups have been removed. Rhamnogalacturonase further exhibited no degrading activity against a polysaccharide fraction (SPS) present in soy-bean isolates in contrast to the commercial enzyme preparation itself which is known to possess activity against SPS (UK Patent 2 115 820). This finding illustrates that the commercial enzyme preparation used is indeed a crude enzyme preparation comprising various enzymes with different activities. In their paper Schols et al (1990a) suggest that rhamnogalacturonase may be useful in studies of the structures of complex pectic polysaccharides, but no further applications are suggested.
No commercial preparations of pure rhamnogalacturonase or comprising a defined and regulated amount of rhamnogalacturonase are presently available. The only method for obtaining an enzyme with RG-ase activity described sofar is the isolation of the enzyme from the aforementioned commercial preparation according to Schols et al. 1990a, a method that is lengthy, requires a large number of steps and is uneconomical.
The object of the present invention therefore is to provide rhamnogalacturonase in a process that is economical and can lead to easy production of pure forms of the desired enzyme. Furthermore the invention is directed at novel polypeptides having RG-ase activity and at novel compositions comprising rhamnogalacturonase in a predetermined amount preferably in an amount greater than 0.01 weight % based on the total weight of polypeptides present in said composition, with more preference for an amount greater than 0.1%. The composition can comprise the polypeptide having rhamnogalacturonase activity alone or in combination with other (hemi)cellulolytic enzymes or pectinases.